Nanoemulsion and Nanoencapsulation of Something

See discussions, stats, and author proﬁles for this publication at: https://www.researchgate.
net/publication/267097796
Nanoencapsulation and nanoemulsion of bioactive compounds to enhance

their antioxidant activity in food
Article · December 2014
CITATIONS READS
5 1,390
3 authors:
Khaled Fahmy Mahmoud Khaled M A Ramadan

National Research Center, Egypt chemistry and biochemistry
13 PUBLICATIONS 9 CITATIONS 8 PUBLICATIONS 15 CITATIONS
SEE PROFILE SEE PROFILE
Ihab S. Ashoush
College of Agriculture, Ain Shams University, Cairo, Egypt
45 PUBLICATIONS 157 CITATIONS
SEE PROFILE
Some of the authors of this publication are also working on these related projects:
natural additives View project
Nanotechnology and Biotechnology View project
All content following this page was uploaded by Ihab S. Ashoush on 23 October 2014.
The user has requested enhancement of the downloaded ﬁle.

International Journal of Food Science
and Technology (IJFST)
ISSN(P): Applied; ISSN(E): Applied
Vol. 4, Issue 3, Dec 2014, 1-21
© TJPRC Pvt. Ltd.
NANOENCAPSULATION AND NANOEMULSION OF BIOACTIVE COMPOUNDS TO

ENHANCE THEIR ANTIOXIDANT ACTIVITY IN FOOD
KHALED F. M1, KHALED M. A. RAMADAN2 & I. S. ASHOUSH3

1
Department of Food Science and Technology, National Research Centere, Dooki, Cairo, Egypt
2
Department of Agricultural Biochemistry, Faculty of Agriculture, Ain-Shams University, Cairo, Egypt
3
Department of Food Science, Faculty of Agriculture, Ain- Shams University, Cairo, Egypt
ABSTRACT
This work aims at investigating the effect of the nanoemulsion and nanoencapsulation delivery systems on the
antioxidant and antimicrobial activity of different bioactive components. Thymol, carvacrol, linalool and eugenol
hydrophobic compounds were encapsulated in dextran of oil-in-water (o/w) nanoemulsions prepared by ultrasonic
homogenization method and stabilized by different types of emulsifier Tween 20, 40 or 80 and distilled water. The results
showed that the reducing power, antioxidant capacity, H 2O2 scavenging and antioxidant activity of un-encapsulated
eugenol was the highest percent compared others essential oils. The highest essential oils nanoencapsulated yield (EY%)
and efficiency (EE%) were observed for eugenol 91.74 and 97.81 %, respectively by using Dex/T 20 treatment.
Nanoencapsulation of eugenol can enhance the aqueous solubility of eugenol at 25, 35 and 45 °C. Thermal stability of
eugenol increased from 63.5 to 143 °C after nanoencapsulation by using Differential Scanning Coliremetry (DSC).
Antioxidant activity of nanoencapsulated eugenol 0.5 mg/ml in linolic acid system compred with BHT activity at different
periods storage were 98.97, 98.42, 98.12 and 97.89 % at 0, 60, 120 and 180 days, respectively. Sensory evaluation of jelly
prepared with different levels of nano-encapsulated eugenol compared to 1% its un-encapsulated showed the Dex/T20
(0.3 %) formation was the most accepted by testers. The antimicrobial activity of nano-encapsulated eugenol was measured
against three different microorganisms, such as Molds & yeasts, coliforms and salmonella. Significance and Impact of the
Study: nanoencapsulation affects positively essential oils main compounds, improves antioxidant activity and retains
antimicrobial activity, enhancing the quality of the oils.
KEYWORDS: Nano-Emulsion, Nano-Encapsulation, Bioactive Compounds, Thymol, Carvacrol, Eugenol, Linalool
INTRODUCTION
Plant phenolic and polyphenolic compounds are widespread components of the human diet found in vegetables,
fruits, soybean, cereals, tea, coffee, red wine and natural herb extracts. The extracts of medicinal plant and culinary herb
sources possess very strong antioxidant activity in vitro. This antioxidant activity is due mainly to phenolic compounds
present in plants (Sahaa et al, 2008). The strong antioxidant activity of the volatile extract of thyme is mostly due to its
main volatile components, thymol and carvacrol. In the case of the volatile extract of basil, eugenol, reported as a major
volatile component, probably contributes significantly to the strong antioxidant activity of the extract. Eugenol showed
high antioxidant activity in the aldehyde/carboxylic acid assay (Teissedre & Waterhouse 2000; and Lee, & Shibamoto
2001).
www.tjprc.org editor@tjprc.org
2 Khaled F. M, Khaled M. A. Ramadan & I. S. Ashhous
Essential oils (EO) are a group of ethereal lipophilic compounds, extracted from herbs and spices such as thyme
(thymol), oregano (carvacrol) and clove (eugenol) (Burt, 2004). EO are volatile and highly aromatic, with strong
antioxidant, antibacterial and antifungal properties (Toure et al, 2007). When applying EO at a level above their solubility
limit, the visible insoluble EO, particularly in beverages, may be a quality defect unacceptable by consumers.
Encapsulation is the most commonly employed approach in the food industry to disperse lipophilic compounds.
In addition, effective encapsulation technologies protect sensitive compounds from degradation, mask unpleasant tastes of
certain compounds, reduce losses due to evaporation, prevent binding or interaction with other food matrix components,
or facilitate controlled release under desired conditions (McClements et al, 2007; de Vos et al, 2010). Proteins and
carbohydrates are frequently studied as matrices to encapsulate lipophilic compounds by spray drying (Charve and
Reineccius 2009; Gharsallaoui et al, 2007; Sliwinski et al, 2003; Zeller et al, 1998), which is a one-step, low-energy and
economical encapsulation method commonly used in the food industry (Adamiec and Kalemba 2006). Specifically relevant
to this study, mixtures of whey protein isolate (WPI) and maltodextrin (MD) have been used to encapsulate avocado
(Bae and Lee, 2008), ginger (Toure et al, 2007), oregano (Baranauskiene, et al, 2006) and caraway (Bylaite et al, 2001)
essential oils. Moreover, conjugation of WPI and MD by dry heating (the Maillard reaction) improves emulsifying
properties of whey protein (Akhtar and Dickinson 2007; Choie et al, 2010). For whey proteinbased encapsulation systems,
heat stability of capsules is an important parameter because manufacturing of many beverage products requires thermal
processing to inactivate spoilage and pathogenic microorganisms, but whey protein is known to aggregate during heating,
especially at increased ionic strength and acidity near isoelectric point when electrostatic repulsion weakens (Bryant and
McClements 1998). Further, to prepare transparent dispersions, nanoparticles with encapsulated lipophilic compounds
typically smaller than 100 nm are required to reduce the scattering of light (Farhang 2009). However, heat stable
transparent dispersions with EO-containing capsules based on whey protein or conjugates have not been reported.
Recently, studies have shown the successful approach of using nanoemulsions to improve stability in food
applications. Tan (2005) and Yuan et al, (2008) prepared EO nanodispersions using high-pressure homogenization and
studied their physicochemical properties. Other applications include the encapsulation of limonene, lutein, omega-3 fatty
acids, astaxantin, and lycopene (Chen et al, 2006), the encapsulation of a -tocopherol to reduce lipid oxidation in fish oil
(Weiss et al, 2009), and the use of nanoemulsions to incorporate essential oils, oleoresins, and oil-based natural flavors into
food products such as carbonated beverages and salad dressings (Ochomogo and Monsalve- Gonzalez 2009).
In the last two decades, nanotechnology has rapidly emerged as one of the most promising and attractive research
fields. The technology offers the potential to significantly improve the solubility and bioavailability of many functional
ingredients.
The high hydrophobicity of some bioactive substances makes them insoluble in aqueous systems, and they
therefore have a poor intake in the body. To improve antioxidants dispersibility in water, for example (Horn and Rieger
2001), and also to increase their bioavailability during gastrointestinal passage (Deming and Erdman 1999), antioxidant
crystals must be formulated, that is, to incorporate them in the fine particles of oil-in-water (O/W) emulsions.
They can be produced using two different approaches: high-energy and low energy methods. High-energy
methods use intense mechanical forces to break up macroscopic phases or droplets into smaller droplets and typically
involve the use of mechanical devices known as homogenizers, which may use high-shear mixing, high-pressure
homogenization, or ultrasonication. In contrast, low-energy methods rely on the spontaneous formation of emulsions under
Nanoencapsulation and Nanoemulsion of Bioactive Compounds to Enhance their Antioxidant Activity in Food 3
specific system compositions or environmental conditions as a result of changes in interfacial properties (McClements and
Li 2010). Spontaneous emulsification is a less expensive and energy-efficient alternative that takes advantage of the
chemical energy stored in the system (Bilbao Sáinz et al, 2010). High-energy methods are effective in reducing droplet
sizes but may not be suitable for some unstable molecules, such as proteins or peptides.
One of the most used low-energy methods is the phase inversion temperature (PIT) method, which is based on the
changes in solubility of polyoxyethylene-type nonionic surfactants with temperature. The surfactant is hydrophilic at low
temperatures but becomes lipophilic with increasing temperature due to dehydration of the polyoxyethylene chains. At low
temperatures, the surfactant monolayer has a large positive spontaneous curvature forming oil-swollen micellar solution
phases (or O/W microemulsions), which may coexist with an excess oil phase. At high temperatures, the spontaneous
curvature becomes negative and water-swollen reverse micelles (or W/O microemulsions) coexist with an excess water
phase. At a critical temperature—the hydrophilic–lipophilic balance (HLB) temperature—the spontaneous curvature is
zero and a bicontinuous microemulsion phase containing comparable amounts of water and oil phases coexists with both
excess water and oil phases.
If the cooling or heating process is not fast, coalescence predominates and polydispersed coarse emulsions are
formed (Ee et al, 2008).
Other low-energy methods that can be used to prepare nanoemulsions are the PIC (phase inversion composition)
method, in which the temperature is maintained constant and the composition is changed (the solvent quality is changed by
mixing two partially miscible phases together). By using the PIC method, different nanomaterials such as colloidosomes
(Dinsmore et al, 2002), cubosomes (Spicer 2004), and microfluidic channels (Xu et al. 2005) have been prepared. Liu et al,
(2006) used polyoxyethylene (PEO) nonionic surfactants for the preparation of paraffin oil-in water nanoemulsions also by
using the PIC method. They reported the preparation of 2.1 Methods of Formation 9 stable nanoemulsions with diameters
ranging from 100 to 200 nm. Surfactants used in this system were a combination of Span 80, a sorbitan monooleate with a
low HLB, and Tween 80, an ethoxilated sorbitan monooelate ester with a high HLB. As these two surfactants possess the
same backbone, they can mix easily, leading to a controlled change in the final HLB.
Another commonly used inversion method is transitional phase inversion (TPI) (Jahanzad et al, 2010). Before TPI
can occur, the surfactants in both phases must diffuse toward the interface, adsorb at the interface, and conform into a
mixed surfactant layer at the optimum conditions. The rate of diffusion of the surfactants depends on many parameters,
including their size, the viscosity of the phase, and the intensity of mixing, etc. For oil-in-water emulsions containing a pair
of water soluble and oil-soluble surfactants, it was found that the addition of the water phase containing the water-soluble
surfactant to the oil phase containing the oil-soluble surfactant may produce very fine emulsions if it is associated with
interfacial tension lowering in the course of addition. The rate of addition of the second phase is of great importance in
achieving an emulsion with the desired properties. This is Nano and Micro Food Emulsions because the dynamic of phase
inversion emulsification is very fast, and the emulsion properties change quickly with further addition, contrary to some
conventional emulsification methods. Therefore, it is important to find and maintain a semi equilibrated state in the course
of emulsification during which sufficient surfactant diffusion/adsorption can occur, and thus droplet rupturing is enhanced.
TPI occurs when the curvature of the oil–water interface gradually changes from positive to negative, passing through a
zero curvature at the inversion point. This is associated with a shift in the surfactant nature from water-soluble to
oil-soluble, or vice versa.
Conventional emulsions, that is, emulsions with microdroplets, are thermodynamically unstable liquid–liquid
dispersions. Emulsions with particle sizes higher than 100 nm are usually prepared by high-energy methods. As in the case
of nanoemulsions, the O/W systems consist of lipid droplets dispersed in an aqueous medium, with each lipid droplet being
surrounded by a thin emulsifier layer. The initial droplet concentration and size distribution of this type of delivery system
can be controlled, as can the nature of the emulsifier used to stabilize the droplets. A careful selection of emulsifier type
enables one to control interfacial properties such as charge, thickness (dimensions), rheology, and response to
environmental stresses (such as pH, ionic strength, temperature, and enzyme activity) (McClements and Li 2010). To form
conventional emulsions, usually a preemulsion with coarse particles, obtained by a mechanical device such as an
Ultra-Turrax, is prepared. This system is further homogenized to obtain a micro droplet emulsion. In general, a variety of
homogenizers are available to prepare emulsions, including high-shear mixers, high-pressure homogenizers, colloid mills,
ultrasonic homogenizers, and membrane homogenizers.
Encapsulation of bioactive compounds represents a feasible and efficient approach to modulate drug release,
increase the physical stability of the active substances, protect them from the interactions with the environment, decrease
their volatility, enhance their bioactivity, reduce toxicity, and improve patient compliance and convenience (Ravi 2000).
Encapsulation of Essential Oils deals with micrometric size capsules, which are used for the protection of the
active compounds against environmental factors (e.g, oxygen, light, moisture, and pH), to decrease oil volatility and to
transform the oil into a powder. Encapsulation in nanometric particles is an alternative for overcoming these problems but
additionally, due to the sub cellular size, may increase the cellular absorption mechanisms and increasing bioefficacy
(Anna et al, 2014).
Carvacrol, limonene and cinnamaldehyde were encapsulated in the sunflower oil droplets of nanoemulsions
prepared by high pressure homogenization and stabilized by different emulsifiers: lecithin, pea proteins, sugar ester and a
combination of Tween 20 and glycerol monooleate. The antimicrobial activity was measured against three different
microorganisms, such as Escherichia coli, Lactobacillus delbrueckii and Saccharomyces cerevisiae. The measured
antimicrobial activity was significantly affected by the formulation of the nanoemulsion, where the different bioactive
compounds were encapsulated. In particular, the effect of the delivery systems on the antimicrobial activity was correlated
to the concentration of the essential oil components in the aqueous phase in equilibrium with the nanoemulsion droplets,
suggesting that the ability of the active molecules to interact with cell membranes is associated to their dissolution in the
aqueous phase. (Francesco et al, 2012).
Although the antimicrobial effects of essential oils are well established and despite the several studies on their
antimicrobial activity (Bakkali et al, 2008; Ben Arfa et al, 2006; Ceylan and Fung, 2004; Helander et al, 1998; Lambert et
al, 2001; Ultee et al, 2000, 2002; Sikkema et al, 1995), it must be highlighted that their mechanisms of action are poorly
understood, even though different theories were proposed. In particular, it appears that the different essential oil
components are characterized by a peculiar mechanism of action, which depends on the nature of the specific bioactive
molecule (Di Pasqua et al, 2007).
The objective of this work was to try increase the efficiency and stability of some essential oils using
nanotechnology (nano-emulsion and nano-encapsulation) techniques on the antioxidant and antimicrobial activity for
application in the food industry.
MATERIALS AND METHODS

Materials
Pure bioactive compounds, Thymol, Euganol, Linalool and Carvacrol were delivered from Sigma – Aldrich
Chemicals.
Methods
Reducing Power
The reducing power of thymol, carvacrol, linalool, and eugenol was determined according to the method of
Oyaizu, (1986). A volume of 0.4 ml of ethanolic solutions with different concentration (0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0
mg/ml) form each tested compound were added to 1 ml potassium ferricyanide 1% and 1 ml phosphate buffer 0.2 M, pH =
6.6. Mixture was incubated at 50 oC for 20 min. Then, trichloroacetic acid 10% was added to mixture, and centrifuged at
650 g for 10 min. Two ml of the supernatant was mixed with 2 ml distilled water and 0.4 ml ferric chloride 0.1% and the
absorbance was read spectrophotometrically( Shimadzu UV-VIS 160 A) at 700 nm. Higher absorbance of the reaction
mixture indicated greater reducing power.
Total Antioxidant Capacity as Equivalents of α-Tocopheroel
The total antioxidant capacity of thymol, carvacrol, linalool and eugenol was determined according to the method
of Prieto et al. (1999). Ethanolic solution with different concentration (10, 20, 30, 40, 50, 70, 100 and 500 ppm) was added
to 3.5 ml reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4mM Ammonium molybdate). Then, the
tubes were capped and incubated in a water bath at 95 oC for 90 min. After cooling to room temperature, the absorbance
was measured at 695 nm against a blank. The antioxidant capacities expressed as equivalents of α-tocopheroel.
The Antioxidant Capacity of Thymol, Carvacrol, Eugenol and Linalool by DPPH Method
The ability of tested compounds to scavenge DPPH radical was determined according to the method of (Hatano,
et al, 1988). The reaction mixture had contained 0.1 ml of DPPH radical solution (5 mM) and different concentration of
tested compounds (from 0.0026 mM to 83 mM). Total volume of the reaction mixture was 3ml. Absorption of DPPH
radical at 515 nm was determined after 10 min against a blank solution that contained only methanol.
% DPPH radical scavenging activity = ((Ac-As)/Ac) ×100
Where: As= absorbance of sample; Ac= absorbance of control (DPPH radical solution in methanol without
sample).
Scavenging of Hydrogen Peroxide
The ability of tested compounds to scavenge hydrogen peroxide was determined according to the method of Ruch
et al, (1989). A solution (20 mM) of hydrogen peroxide was prepared in ethanol 95%. Hydrogen peroxide concentration
was determined spectrophotometrically from absorption at 230 nm by using the molar absorptive of 81M -1 cm-1. Ethanolic
solution of each sample with different concentration 10, 20, 40, 60, 80, 100, 120,140 ppm was added to a hydrogen
peroxide solution. Absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank.
% scavenging of hydrogen peroxide effect = (As/Ac -1) ×100
Where: As= absorbance of sample, Ac= absorbance of control (hydrogen peroxide solution in ethanol without
sample).
Antioxidant Activity in a Linoleic Acid System
An antioxidant activity assay was carried out by using the linoleic acid system Osawa and Namiki (1981). Tested
compounds in ethanolic solution with different concentration (125, 250, 375,500 ppm) were added to 2 ml ethanolic
solution of linoleic acid 0.2 M. Then, the total volume was adjusted to 4 ml with ethanol. And the reaction mixture
incubated at 40oC. the degree of oxidation was measured according to the thiocyanate method Mitsuda et al, (1996) by
adding 0.1 ml reaction mixture , 0.1 ml ammonium thiocyanate 30% , 3 ml solvent (n butanol :ethanol 1:1) and 0.1 ml
ferrous chloride (20 mM in 3.5% HCl). After mixing was stirred for 3 min, the absorbance measured at 500 nm, and the
percentage of antioxidant activity was calculated from the following equation:
Antioxidant activity %= 100 - [(absorbance of sample / absorbance of control) ×100]
All tests and analyses were run in triplicate and averaged
Preparation of Bioactive Nanoemulsions
Oil-in-water (o/w) emulsions were prepared according to Helder et al, (2011) with some modifications.
Briefly: 0.03% (w/w) bioactive compounds (higher than 97 % purity) was dissolved at 40 ºC in n-hexane (95% purity).
This organic solution was then added to an aqueous solution containing 0.5 % (w/w) Tween (20, 40 and 80) (97%, Sigma,
USA) in distilled water. The organic/aqueous phase volume ratio of 1: 9 was used. The emulsions were homogenized using
an ultrasonicator homogenizer (Homogenizer PRO 400PC). The n-hexane was then removed from the fine emulsion by
rotary evaporation. In order to simulate industrial production environment, no special care was taken to avoid contact with
oxygen.
The nanoemulsions obtained were stored at 4 ºC in the absence of light, during the evaluation period, reproducing
eventual commercial conditions.
 2 % dextran was added to 0.5 % (w/w) Tween 80 and then 100 ml distilled water and stirring (Dex/T 80).
 2 % dextran was added to 0.5 % Tween 40 and then 100 ml distilled water (Dex/T 40).
 2 % dextran was added to 0.5 % Tween 20 and then 100 ml distilled water (Dex/T 20).
Determination of Nano-Encapsulation Efficiency (NE) of the Clove Powder
The encapsulation efficiency was calculated as the amount of eugenol present in the nano-capsules compared with
the eugenol initially used in their production. The nano-capsule was completely destroyed in accordance to the procedure
reported by Robert et al, (2010), with little modifications. 5 ml of the encapsulated solution was added to 10 ml methanol.
The dispersion was agitated using a vortex (1 min). After vortexing, the samples were allowed to rest for 5 min while
protected from light and then in an Ultrasonicator (M/s Ika, Germany) for 30 min at 1000 rpm, then centrifuged at 112,000
g for 5 min and finally filtered. The supernatant was analyzed for bioactive compounds such as total eugenol content, total
phenolic content and antioxidant activity.
The nanoencapsulation efficiency (NE) was calculated according to Eq. (1) using the results from total bioactive
compounds and theoretical bioactive compounds (Ahmed et al, 2010).
(1)
Encapsulation Yield
The encapsulation yield (EY) was calculated according to Eq. (2) as the ratio of the mass of nanocapsules
obtained after ultrasonic possessing to the total mass of the initial substances added before encapsulation Duduku et al,
(2012).
(2)
Oxidation Study of Bioactive Compounds Encapsulated forms by DSC
DSC was furthermore employed to study the thermo-oxidation stability of the samples. Samples of pure terpenes
as well as their complexes with B-CD and their encapsulated forms in MS containing around 2 mg of terpenes were placed
in 40-μL aluminum pans having a hole in their lid. The specimens were heated in pure oxygen atmosphere from room
temperature to 120 ◦C at 90 ◦C/min, remained at 120 ◦C for 1 min, to ensure a uniform temperature distribution within the
sample, and then heated up to 420 ◦C at 10 ◦C /min.
Application and Preparation of Jelly
Jelly was prepared in laboratory by adding different levels of eugenol nanoencapsulated i.e. 0.10, 0.20, 0.30, 0.40
and 0.50% w/w in laboratory using the traditional procedure. The formulation of Jelly was contained the ingredients of
sucrose, gelatin, citric acid, flavoring agent, sodium benzoate and potassium citrate, ascorbic acid and antioxidant pigment
were 84.0, 15.0, 0.20, 0.10, 0.10, 0.10 and 0.1-0.5 %, respectively.
Jellies were wrapped by polyethylene and aluminum foil and packed in carton bags and were stored at room
temperature 25±5ºC. The control of jelly was prepared with 0.10% synthetic color (carmine) according to Attia et al,
(2013)
Phase Solubility Studies
Phase solubility studies were carried out as described by Higuchi and Connors (1965) and Mourtzinos et al,
(2008). Briefly, an excess amount of eugenol encapsulated was mixed with an aqueous solution of dextran of increasing
concentrations (0 to 0.015 M). A laboratory shaker was used and the samples were equilibrated for 2 days at 25, 35, and 45
ºC at gas-tight bottles. Then the samples were filtered with a syringe through 0.45-µm pore diameter PVDF filters, and the
amount of eugenol encapsulated in solution was determined spectrophotometrically by measuring the absorbance at 280.2
nm. The experiments were carried out in triplicate at each temperature.
The stability constants, KC, of the reaction of dextran toward a complex formation were calculated from the
straight-line portion of the phase solubility diagram according to the Higuchi–Connors equation:
Microbiological Analyses
For microbiological evaluation, the following three analyses were performed: yeast and mold counting, coliforms
at 45°C and Salmonella sp. research using the techniques outlined in the current legislation, RDC # 12 (BRASIL, 2001).
Sensory Evaluation
An acceptance test of the jelly samples was carried out at the Sensory Analysis Laboratory of the Food Science
and Technology Department, National Research Center (NRC) with the participation of 41 volunteer members of the NRC
community from both sexes and of all ages. Volunteers were selected based on their interest and availability to take part in
the tests. Samples were evaluated through tests of preference and intention to purchase in accordance with NBR
12806/1993 (ABNT, 1993). The testers assessed the samples using a 9-point structured hedonic scale, as follows:
1 - disliked very much; 2 - disliked quite a lot; 3 - fairly disliked; 4 - slightly disliked; 5 - neither liked nor disliked;
6 - slightly liked; 7 - fairly liked; 8 - liked quite a lot; 9 - liked very much. The samples were marked with random three
digit numbers and served to testers in individual booths under white light. Plain mineral water and biscuit means cookie
were provided for rinsing of the palate between sample evaluations.
Statistical Analyses
Experimental results were mean ± S.D. of three parallel measurements and analyzed by SPSS 10 (SPSS Inc.
Chicago, IL). Differences between means were determined using Tukey multiple comparisons and least significant
difference (LSD). Correlations were obtained by Pearson correlation coefficient in bivariate correlations. P values <0.05
were regarded significant.
RESULTS AND DISCUSSIONS

Antioxidant Activity of the Basil and Essential Oils Major Components
Reducing Power
The reducing power of thymol (T), carvacrol (C), linalool (L), and eugenol (E) was measured by the ferricyamide
method. Different concentrations of thymol, carvacrol, linalool and eugenol 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml were
investigated. Higher absorbance at 700 nm of the reaction mixture indicated greater reducing power. Table 1 represents the
reducing power of T, C, L and E measured as absorbance at 700 nm. Table 1 reveals that T, C, E exhibited a reducing
power, but to varying degrees while L has no significant reducing power. A positive correlation has been observed between
concentrations and reducing power for T, C, E especially E. There is a significant difference among the tested compounds
in their reducing power.
Table 1: Reducing Power of Thymol, Carvacrol, Linalool and Euganol as Absorbance at 700 nm
Absorbance at 700 nm
Concentration
Ascorbic
(mg/ml) Thymol Carvacrol Linalool Eugenol
Acid
0.05 0.36±0.01 0.23±0.02 0.04±0.01 0.96±0.03 0.83±0.01
0.1 0.66±0.02 0.53±0.02 0.09±0.01 1.64±0.02 1.36±0.11
0.2 1.05±0.01 0.87±0.01 0.14±0.01 2.15±0.05 1.68±0.06
0.4 1.48±0.20 1.26±0.02 0.18±0.01 2.73±0.03 1.87±0.03
0.6 1.64±0.06 1.41±0.04 0.20±0.01 2.91±0.07 2.03±0.05
0.8 1.73±0.04 1.56±0.06 0.21±0.01 3.37±0.13 2.05±0.12
1.0 1.78±0.05 1.68±0.03 0.22±0.01 3.92±0.11 2.07±0.07
The reducing power of E was higher than T than C in the same order, we have notice that the reducing power of E
was about 7-fold the reducing power of T and C.
Politeo, et al. (2007) reported that the highest reducing power (FRAP) was showed samples of pure eugenol.
The reducing power of the volatile aglycones is comparable, but less than the reducing power of essential oil and BHT.
A hierarchy in the reducing capacity of samples could be observed as well: eugenol > BHT > basil essential oil > basil
volatile aglycones. Among the compounds identified in the essential oil and volatile aglycones from basil, eugenol was
considered the main contributor of the antioxidant capacity. The comparison of the antioxidant capacity of pure eugenol
and the total essential oil at corresponding to equivalent mass of eugenol in total essential oil (EC50 = 0.099 g/l) was
showed very similar antioxidant capacities for both in all concentration range.
The Antioxidant Capacity of Thymol, Carvacrol, Eugenol and Linalool by Phosphomolybdenium Method
The antioxidant capacity of T, C, E and L by phosphomolybdenium method were determined and expressed as
equivalents of α- tocopherol (nmol/g oil). Different concentrations of T, C, E and L 10, 20, 30, 40, 50, 70, 100 and 500
ppm were measured.
Table 2: The Antioxidant Capacity of Thymol, Carvacrol, Linalool and Eugenol

by Phosphomolybdenium Methods as Equivalents of α-Tochopherol
Conc. Antioxidant Capacity %
(ppm) Thymol Carvacrol Linalool Eugenol
10 00.00 00.00 13.52 27.83
20 00.00 00.00 51.67 76.65
30 00.00 00.00 75.02 99.52
40 00.00 00.00 81.34 100.0
50 4.65 1.71 82.19 100.0
70 12.11 8.31 82.28 100.0
100 28.26 24.58 83.09 100.0
500 35.74 31.91 84.56 100.0
The obtained results showed that E, L, T and C have an antioxidant capacity, but with different degrees as shown
in Table 2. The antioxidant capacity of all tested compounds increased with the concentrations. There was a significant
difference among the tested compounds in their antioxidant capacity. Ranking of increasing the antioxidant capacity is E >
L > T > C in all tested concentration. We have notice that the antioxidant capacity of E and L was very high compared to
the antioxidant capacity of T and C and this result was in good agreement with the previous obtained result Islam et al,
(2006).
The antioxidant capacity of L was very high but its reducing power is very low. So, there was a very high
difference between the reducing power and the antioxidant capacity of L. linalool is a monoterpen which has an allelic
structure, its structure can be reacted with H2SO4 which present in the reaction mix in the antioxidant capacity and a
sulfunyl compound will form. So that, sulfonyl compound reacts and give a high antioxidant capacity. The difference
between the reducing power and the antioxidant capacity of L related to the reaction conditions because of the reaction
mixture of antioxidant capacity contained H2SO4 0.6  and the reaction mix was heated at 95C for 90 min. so, its false
antioxidant capacity. This mechanism can explain according to March (1968). On the other hand, the reducing power and
antioxidant capacity of E were the highest activity. So, that, BO has a high reducing power and antioxidant capacity
because of its containing of E. from the obtained results, T and C have low reducing power and antioxidant capacity.
In addition, to has a low reducing power and antioxidant capacity. As previously mentioned the reducing power and the
antioxidant capacity of E is the highest activity. Mechanism of antioxidant action is reducing power and antioxidant
capacity of E could be explained according to Ou et al. (2002)
The Percent of H2O2 Scavenging Activity of Thymol, Carvacrol, Linalool and Eugenol
The percent of H2O2 scavenging activity of T, C, L and E was determined at different concentrations 10, 20, 40,
60, 80, 100, 120 and 140 ppm. As shown in Table 3, all tested compounds exerted a concentration-dependent scavenging
of H2O2 except L. The concentration of H2O2 in the systems containing T, C and E samples dropped very sharply during
the initial 10 min period of the assay especially in E samples. The percent of H2O2 scavenging activity of T, C and E
increased in the following order E > T > C at all tested concentrations. There was a significant difference among all tested
compounds in the percent of H2O2 scavenging activity.
Table 3: The Percent of H2O2 Scavenging Activity of Thymol, Carvacrol, Linalool and Eugenol
% H2O2 Scavenging Activity
Concentration
Ascorbic
mg/ml Thymol Carvacrol Linalool Eugenol
Acid
0.01 35.63±0.02 4.27±0.02 0.00±0.00 18.45±0.02 28.21±0.01
0.02 39.51±0.01 18.94±0.04 0.00±0.00 53.72±0.03 52.31±0.01
0.04 53.82±0.03 25.18±0.05 0.00±0.00 78.51±0.12 77.47±0.08
0.06 60.04±0.11 33.56±0.03 0.00±0.00 96.38±0.23 98.58±0.12
0.08 67.93±0.21 43.28±0.07 0.00±0.00 99.35±0.21 98.59±0.11
0.1 76.53±0.23 56.46±0.13 0.00±0.00 100±0.11 98.60±0.10
0.12 96.38±0.08 82.41±0.16 0.00±0.00 100±0.04 98.60±0.10
0.14 96.42±0.07 89.23±0.10 0.00±0.00 100±0.01 98.60±0.09
The obtained results showed that the percent of H2O2 scavenging activity of E was the highest percent. As shown
in Table 3, the percent of H2O2 scavenging of E was 99.35 % and 100% at concentrations 0.08 and 0.1 mg/ml respectively,
while L has no H2O2 scavenging activity related to the phenolic structure where the phenolic compounds (E, T and C) have
H2O2 scavenging activity while the non-phenolic compound (L) has no H2O2 scavenging activity. Mechanism of H2O2
scavenging activity could be explained according to Wettasinghe and Shahidi (1999) as shown in Part I. Since the phenolic
compounds (E, T and C) are good hydrogen atom donors, they may accelerate the conversion of H2O2 to H2O.
Aysun and Ayse (2011) reported that the essential oil showed a higher membrane-protective effect than thymol
and carvacrol. The three major constituents of the essential oil were linalool (50.53%), carvacrol (24.52%), and thymol
(15.66%), respectively.
The Antioxidant Activity of Thymol, Carvacrol, Linalool and Eugenol in LInoleic Acid System
The antioxidant activity of T, C, L and E were determined in linoleic acid system and compared with BHT
activity. Different concentrations used in determination 125, 250, 375 and 500 ppm for the tested compounds and 200 ppm
for BHT. The linoleic acid and tested compounds samples mixtures were incubated at 40ºC for 3 days. Every day, samples
were taken for determination of the antioxidant activity.
Table 4: The Antioxidant Activity of Thymol, Carvacrol, Linalool and

Eugenol in Linolic Acid System Compared with BHT Activity
Conc. % Antioxidant Activity
(mg/ml) Thymol Carvacrol Linalool Eugenol BHT 0.2 mg/ml
0.125 65.31 48.05 72.84 91.05 88.17
0.250 67.49 50.91 75.27 93.52 90.23
0.375 69.82 53.57 79.43 95.16 92.74
0.500 71.33 58.40 81.61 97.48 95.11
All tested compounds exhibited an antioxidant activity, but to various degrees except L as shown in Table 4.
A positive correlation has been observed between concentrations (for T, C and E) and antioxidant activity. There is a
significant difference among the tested compounds in their antioxidant activity. The antioxidant activity of the tested
compounds decreased in the following order: T > E > C > BHT > L. The best antioxidant activity of all tested compound
(T, C and E) observed from 0.5 mg/ml concentrations.
Thymol is the best antioxidant activity in all concentration then carvacrol then Eugenol then BHT then linalool.
The antioxidant activity of all tested compound, increased with concentrations except linalool. So that, the best
concentration of thymol, carvacrol and eugenol was 0.5 mg/ml in the antioxidant activity. The antioxidant activity of L was
a negative value, the negative value. The negative value of the antioxidant activity increased with concentrations.
The negative antioxidant activity of L could be explained because of the peroxide value of L samples was higher than the
peroxide value of control where that the calculation equation was: 100 – [(absorbance of sample/absorbance of control) X
100]. So that we have to notice that L has a pro-oxidant effect.
The results were hugher that that reported by Aysun and Ayse (2011) the 6 major components of the essential oil
were linalool (50.53%), carvacrol (24.52%), thymol (15.66%), cymene (2.68%), terpinen-4-ol (2.06%), and gamma-
terpinene (1.41%), respectively.
Characterization of Nanoemulsion and Nanoencapsulation of Bioactive Compounds
Nanoemulsions are not only used to change the texture, flavor, and appearance of foods, but they are increasingly
used to delivery biologically functional components that are lipophilic such as antioxidants, flavors, and nutraceuticals.
Traditionally, emulsions could only be manufactured on a commercial scale with droplets in the nano-size range 10-100
nm) (Friberg et al, 2004; McClements, 2005a; Leal-Calderon et al, 2007).
Encapsulation of essential oils at the nanoscale represents a viable and efficient approach to increasing the
physical stability of the bioactive compounds, protecting them from the interactions with the food ingredients.
Encapsulation of functional ingredients has been shown to reduce ingredient interactions and allow for better control over
mass transport phenomena and chemical reactions. Encapsulation of biologically active components such as nutraceuticals
may also improve bioactivity, e.g. by improving adsorption and uptake of components during digestion. In the case of food
antimicrobials, encapsulation may increase the effectiveness of concentration of bioactive compounds in areas of the food
system where target microorganisms are preferentially located (Jochen et al, 2009).
Nano-Encapsulation Yield (EY)
Figure 1 showed that the values EY of the essential oils nanoencapsulated. High EY values for nanocapsules
obtained by treatment Dex/T20 of eugenl was 91.74 %
Figure 1: Nano-Encapsulation Yield of of Thymol, Carvacrol, Linalool and Eugenol in Dextran
This encapsulation yield was higher than that reported by Madhu and Prabhu, (1985) and Frank and
Hans-Joachim, (2007) being 50 %.
Encapsulation Efficiency (%EE)
The results in Figure 2 showed higher values for %EE for the eugenol encapsulated by Dex/T20 was 97.81 %
Figure 2: Nano-Encapsulation Efficiency of Thymol, Carvacrol, Linolool and Eugenol in Dextran
This nano-encapsulation efficiency of eugenol and linalool and thymol were 97.81, 83.79 and 71.51 %,
respectively, but low level in carvacrol. This result was higher than that reported by Dipan and paramita (2013) of the
bioactive compounds such as eugenol, phenolic compounds and antioxidants are in the range of 60–65%.
The nano-encapsulated eugenol was choosen for subsequent studies due to its higher yield and efficiency.
Solubility of the Eugenol Nano-Encapsulated
Essential oils are slightly soluble in water and impart their odor and taste to the water. The results in Figure 3
showed that the solubility of eugenol nanoencapsulated was Increases with temperature and was higher at 45 °C as a result
of a concentration of dextran 0.01 Molar. This result was agreement with that reported by Regiane et al, (2013).
The physical properties of the nanoencapsulated eugenol related to its ease of dispersion in an aqueous solution
include moisture content and respective particle porosity, and the instantanization properties (wetting, dispersibility,
and solubility). These properties are influenced by the nature of the feed (solids content, viscosity, and temperature) as
shown in Figure 4.
Figure 3: Phase Solubility Study of Nano-Encapsulated Eugenol with Dextran in Water at 25, 35, and 45 ºC
Numerous materials, such as dextran (encapsulating agents), are available for encapsulation of essential oils and
application in foods. Dextran is used by the filer industry in applications protecting the core material from oxidation and
volatilization. Moreover, it exhibits high solubility and low viscosity in aqueous solution when compared to other
hydrocolloid dextran which facilitates the spray drying process. The objective of this study was to evaluate the influence of
operational conditions on the properties of nanoencapsulated eugenol essential oil.
Figure 4: Time Related Release of Dextran Nano-Encapsulated Eugenol in Water
Thermal Stability of Nano Eugenol Capsules DSC (Differential Scanning Coliremetry)
The thermal stability of the eugenol nanocapsulated was determined by using Differential Scanning Coliremetry
Figure 5a,b. Higher exothermal peaks still occurred at much higher temperature 143 °C in Figure 5a after nanoencapsulation
of eugenol compared to the initial eugenol before encapsulated was 63.5 °C. This result indicated that capsulation process
caused oxidation prevention and increased the thermal stability of eugenol.
a b
a b
Figure 5: DSC Thermogram of Eugenol before (a) and After Nanoencapsulated

(b) Transmission Electron Microscopy (TEM) of Eugenol Nanoencapsulated
Figure 6 showed TEM micrographs of the investigated models. The particles of the observed sample differ in size
and shape. The morphology of eugenol before encapsulation was obtained by using transmission electron microscopy with
diameter range from 4.2 to 9.8 nm in Figure 6a but was 3.9 to 4.7 nm in Figure 6b
Figure 6: Morphology of Eugenol after Nanoemulsion (a) and Nanoencapsulation

(b) by Transmission Electron Microscopy
Storage Stability of Nano-Encapsulated Eugenol
The results in Table 5 showed that the antioxidant activity of eugenol nano-encapsulated was stable in long-term
180 days storage at 4 °C. However, the loading capacity of eugenol un-capsules is relatively low. To address this issue,
the loss of turmeric oil in key steps in the process was quantified and these data may facilitate improvements in the
procedure to produce an increased loading capacity.
Table 5: Antioxidant Activity of Nano-Encapsulated Eugenol (Dex/T20) in Linolic

Acid System Compared with BHT Activity at Different Periods Storage
Antioxidant Activity of Nano-
Encapsulated Eugenol (%) After
Conc. (mg/ml) Storage
60 120 180
0 Day
Days Days Days
cap. 93.82 93.63 93.25 92.94
0.125
Un-cap. 91.05 86.25 82.74 79.16
cap. 95.24 95.01 94.83 94.62
0.250
Un-cap. 93.52 88.31 85.23 82.77
cap. 97.30 97.13 96.81 96.53
0.375
Un-cap. 95.16 92.06 89.12 86.81
cap. 98.97 98.42 98.12 97.89
0.500
Un-cap. 97.48 95.25 92.61 90.16
BHT 88.17 88.17 88.17 88.17
In order to retain their biological activity and minimize the impact on the organoleptic properties of foods where
they are incorporated, essential oil constituents, like other bioactive compounds, need to be protected from the interaction
with other food ingredients and from environmental stresses experienced during food manufacturing and storage
(McClements, 1999).
Sensory Evaluation of Jelly
Sensory properties of jelly prepared with adding different levels of eugenol nanoencapsulated compared with
control (eugenol 1 % without nano-encapsulation) are given in Table 6. Analysis of variance showed mostly significant
differences in color, taste, odor and overall acceptability for jelly as control by different levels of eugenol encapsulated in
the range 0.1 to 0.6 %. The addition of eugenol encapsulated with different levels significantly affected color, taste, odor
and overall acceptability.
In general, consumer perception has been that treatment as favorable in terms of the general acceptance of the
sample is added by a ratio of 0.3 % eugenol nanoencapsulated was 9.6 %. Addition of the particles of encapsulated
bioactive compounds at amounts up to 5.0% in bread formulation seemed reasonable in terms of general quality parameters
and sensory properties (Burce et al, 2013).
Table 6: Sensory Evaluation of Jelly Prepared with Different Levels of

Nano-Encapsulated Eugenol Compared to 1 % Eugenol Untreated
Parameter
Treatments Overall
Color Taste Odor
Acceptability
Control * 9.80±0.83 9.80±0.79 9.70±0.79 9.70±0.82
0.1 % 7.40±0.71 7.10±0.57 7.20±0.63 7.20±0.65
0.2 % 8.40±0.32 8.20±0.62 8.30±0.71 8.30±0.74
0.3 % 9.80±0.79 9.70±0.81 9.60±0.92 9.60±0.81
0.4 % 8.50±0.63 8.30±0.75 8.30±0.74 8.30±0.77
0.5 % 7.10±0.56 7.00±0.49 7.00±0.61 7.00±0.52
0.6 % 7.00±0.53 6.50±0.47 7.00±0.60 6.50±0.46
* control (eugenol 1 % without nano-encapsulation), LSD: nd (no detected)
Sensory evaluation was applied for the development and improvement of products, quality control and studies on
process. Table 6 shows the average scores for the sensory evaluations. The Dex/T 20 (0.3 %) formulation was the most
accepted by testers, receiving the largest number of indications for the responses ‘liked quite a lot’ and ‘liked very much.’
The Dex/T20 (0.3 %) formulation was the least accepted, receiving the largest number of responses ‘disliked very
much’, for a product to be considered acceptable in terms of its sensory properties; it needs to have a minimum 96% level
of approval.
Microbiological Analysis
The microbiological characteristics observed for the jelly without addition of nano-encapsulated antioxidant
compared to the jelly with addition of nano-encapsulated antioxidant after three months of refrigerated storage are shown
in Table 7.
Table 7: The Microbiological Characteristics of the Jelly with or

without the Addition of Nano-Encapsulated Antioxidant
Control: is jelly without antioxidant; cfu g-1: colony forming units/g
The jelly maintained its microbiological quality during storage and was well within the requirements set forth by
ANVISA. The nano-encapsulated eugenol showed a remarkable inhibitory activity against the growth of jelly
microorganisms, especially Molds & yeasts and no detected salmonella in any samples. More interestingly, the extract
could inhibit strongly the growth of Coliforms, which is regarded as a major etiological concern in gastro-duodenal
disorders. Therefore, the nano-encapsulated of bioactive compounds can be used as a natural source of antimicrobial and
antioxidant agents to preserve foodstuffs against a range of food related microorganisms (Rima et al, 2013).
CONCLUSIONS
Nano-encapsulation offers great advantage for an improved stability of bioactive compounds in certain food
formulations. Jelly as a staple food is considered to be a useful model to develop new generation of functional products,
and to test the stability of nano-encapsulated bioactive compounds. In this study, bioactive compounds were encapsulated
with dextran and added to jelly formulation to test their stability during the process. The results revealed that
nano-encapsulation significantly improved the thermal stability of eugenol at elevated temperatures applied during process.
Addition of the particles of encapsulated bioactive compounds at amounts up to 0.02 % in jelly formulation seemed
reasonable in terms of general quality parameters and sensory properties. Presence of bioactive compounds in a
formulation may bring important changes in the chemical reactions occurring in food during processing. The most
bioactive compounds bear functional groups that may react with other compounds in food. These reactions may lead to the
formation of processing contaminants especially in products in which processing conditions are severe. Therefore,
prevention of the reactivity of bioactive compounds through their nano-encapsulation may also help preventing certain
food safety risks
REFERENCES
1. Adamiec J. and Kalemba D. (2006). Analysis of microencapsulation ability of essential oils during spray drying.
Drying Technology, 24(9): 1127-1132.
2. Ahmed M. G, Satish K. B. P, Kiran K. G. B. (2010). Formulation and evaluation of gastric-mucoadhesive drug

delivery systems of captopril. Journal of Current Pharmaceutical Research, 2: 26–32.
3. Akhtar M. and Dickinson E. (2007). Whey protein-maltodextrin conjugates as emulsifying agents: An alternative
to gum Arabic. Food Hydrocolloids, 21: 607-616.
4. Anna R. B, Clizia G, Benedetta I, Chiara R, Fabio F. and Maria C. B. (2014). Essential Oils Loaded in
Nanosystems: A Developing Strategy for a Successful Therapeutic Approach. Evidence-Based Complementary
and Alternative Medicine, Hindawi Publishing Corporation, Vol. 2014, Article ID 651593, 14 pages,
http://dx.doi.org/10.1155/2014/651593
5. Attia G. Y, Moussa M. E. M. and Sheashea E. R. (2013). Characterization of red pigments extracted from red
Beet (beta vulgaris, l.) and its potential uses as Antioxidant and natural food colorants. Egypt. J. Agric. Res, 91(3):
1095-1110.
6. Aysun O. and Ayse E. (2011). A comparative evaluation of antioxidant and anticancer activity of essential oil
from Origanum onites (Lamiaceae) and its two major phenolic components. Turk Journal Biology. 35: 735-742.
7. Bae E. K. and Lee S. J. (2008). Microencapsulation of avocado oil by spray drying using whey protein and
maltodextrin. Journal of Microencapsulation, 25(8): 549-560.
8. Bakkali, F, Averbeck, S, Averbeck, D, Waomar, M, (2008). Biological effects of essential oils – a review.
Food and Chemical Toxicology 46, 446–475.
9. Baranauskiene R, Venskutonis P. R, Dewettinck K. and Verhé R. (2006). Properties of oregano

(Origanum vulgare L.), citronella (Cymbopogon nardus G.) and marjoram (Majorana hortensis L.) flavors
encapsulated into milk protein-based matrices. Food Research International, 39(4): 413-425.
10. Ben Arfa, A, Combes, S, Preziosi-Belloy, L, Gontard, N, Chalier, P, (2006). Antimicrobial activity of carvacrol
related to its chemical structure. Letters in Applied Microbiology 43, 149–154.
11. Bilbao Sáinz C, Avena Bustillos R. J, Wood D. F, Williams T. G, Mchugh T. H. (2010). Nanoemulsions prepared
by a low-energy emulsification method applied to edible films. J Agric Food Chem, 58: 11932–11938.
12. BRASIL (2001). ANVISA-Agência Nacional de Vigilância Sanitária. Ministério da Saúde. Resolução da diretoria
colegiada da secretaria de vigilância sanitária n. 12. Diário Oficial da União, Brasília: Ministério da Saúde.
13. Bryant C. M, and McClements D. J. (1998). Molecular basis of protein functionality with special consideration of
cold-set gels derived from heat-denatured whey. Trends Food Sci. Technol, 9: 143–151.
14. Burce A. M, Vural G, and Eyal S. (2013). Nano-encapsulation improves thermal stability of bioactive compound
omega fatty acids and silymarin in bread. Agro Food Indusrty Hi Tech, 24(3): 62-65.
15. Burt S. (2004). Essential oils: their antibacterial properties and potential applications in foods – a review.
International Journal of Food Microbiology 94, 223–253.
16. Bylaite E, Venskutonis P. R. and Mapdpieriene R. (2001). Properties of caraway (Carum carvi L.) essential oil
encapsulated into milk protein-based matrices. European Food Research and Technology, 212(6): 661-670.
17. Bylaite E, Venskutonis P. R. and Mapdpieriene R. (2001). Properties of caraway (carum carvi l.) essential oil
encapsulated into milk protein-based matrices. European Food Research and Technology, 212(6): 661-670.
18. Ceylan E. and Fung D. Y. C. (2004). Antimicrobial activity of spices. Journal of Rapid Methods and Automation
in Microbiology 12, 1–55.
19. Charve J. p. and Reineccius G. A. (2009). Encapsulation performance of proteins and traditional materials for
spray dried flavors. Journal of Agricultural and Food Chemistry, 57(6): 2486-2492.
20. Chen H, Weiss J, and Shahidi F. (2006). Nanotechnology in nutraceuticals and functional foods. Food Technol,
60:30–36.
21. Choi M. J, A. Soottitantawat, O. Nuchuchua, S.-G. Min, and U. Ruktanonchai, (2010). “Physical and light
oxidative properties of eugenol encapsulated by molecular inclusion and emulsiondiffusion method,” Food
Research International, 42(1): 148–156.
22. De Vos P, Faas M. M, Spasojevic M. and Sikkema J. (2010). Encapsulation for Preservation of Functionality and
Targeted Delivery of Bioactive Food Components. International Dairy Journal, 20: 292-302.
23. Deming D. M, and Erdman J. W. Jr (1999). Mammalian carotenoid absorption and metabolism. Pure Appl. Chem,
71: 2213–2223.
24. Di Pasqua R, Betts G, Hoskins N, Edwards M, Ercolini D, and Mauriello G, (2007). Membrane toxicity of
antimicrobial compounds from essential oils. Journal of Agricultural and Food Chemistry 55, 4863–4870.
25. Dinsmore A. D, Hsu M. F, Nikolaides M. G, Marquez M, Bausch A. R, and Weitz D. A. (2002). Colloidosomes:
selectively permeable capsules composed of colloidal particles. Science, 298: 1006-1009.
26. Dipan C. and paramita B. (2013). Comparative evaluation of the antioxidant efficacy of encapsulated and
un-encapsulated eugenol-rich clove extract in soybean oil: shelf-life and frying stability of soybean oil. Journal of
Food Engineering, 117: 545-550.
27. Duduku K, Kong B. H, Rosalam S, Anisuzzaman S. M. and Rajesh N. (2012). Spray drying of Morinda Citrifolia
L. and Beta Vulgaris L. fruit extract and its synergistic effect. International Journal of Chemical Engineering and
Applications, 3(6): 380-384.
28. Ee S. L, Duan X, Liew J, and Nguyen Q. D. (2008). Droplet size and stability of nano-emulsions produced by the
temperature phase inversion method. Chem. Eng. J, 140: 626–631.
29. Farhang B. (2009). Nanotechnology and applications in food safety. In: Gustavo, B.-C, Alan, M, David, L,
Walter, S, Ken, B. and Paul, C, editors. Global issues in food science and technology. San Diego: Academic
Press. p 401-410.
30. Francesco D. Marianna A, Mariarosaria V, and Giovanna F. (2012). Design of nanoemulsion-based delivery
systems of natural antimicrobials: Effect of the emulsifier. Journal of Biotechnology, 159: 342– 350.
31. Frank A. E. and J. Hans-Joachim (2007). Immobilization of dextranase from Chaetomium erraticum. Journal of
Biotechnology, 131: 440–447.
32. Friberg S, Larsson K. and Sjoblom J. (eds.) (2004). Food Emulsions. New York: Marcel Dekker.
33. Gharsallaoui A, Roudaut G, Chambin O, Voilley A. and Saurel R. (2007). Applications of spraydrying in
microencapsulation of food ingredients: An overview. Food Research International, 40(9): 1107-1121.
34. Hatano T, Kagawa H, et al, (1988). Two new flavonoids and other consitituents in licorice root; their relative
astringency and radical scavenging effects. Chem. Pharm. Bull, 36: 2090-2097.
35. Helander I. M, Alakomi H. L, Latva-Kala K, Mattila-Sandholm T, Pol I, Smid E. J, Gorris L. G. M, and Von
Wright A. (1998). Characterization of the action of selected essential oil components on gram-negative bacteria.
Journal of Agricultural and Food Chemistry 46, 3590–3595.
36. Helder D. S, Miguel C, Bartolomeu W. S. S, Clara R, Maria C, Mafalda C. Q, Jane S. R. C, Maria G. C, and
António A. V. (2011). Nanoemulsions of β-carotene using a high-energy emulsification–evaporation technique. J.
Food Eng, 102: 130–135.
37. Higuchi T. and Connors K. A. (1965). Phase solubility technique. Adv Anal Chem Instrum, 4: 117–212.
38. Horn D, and Rieger J. (2001). Organic nanoparticles in the aqueous phase—theory, experiment, and use.
Angewandte Chemie, 113:4460–4492.
39. Islam M. M, Hossain M. M, Haider M.G, Chowdhury E. H. and Kamruzzaman M. (2006). Seroprevalence and
pathological studyof Salmonella infections in layer chickens andisolation of causal agents: In Proceedings of the
5th International Poultry show and seminarfrom 01-03 march 2007, held in BangladeshChina Friendship
Conference Centre (BCFCC), Sher-e-Bangla Nagar, Dhaka, Bangladesh. 9-15.
40. Jahanzad F, Josephides D, Mansourian A, and Sajjadi S. (2010). Dynamics of transitional phase inversion
emulsifi cation: effect of addition time on the type of inversion and drop size. Ind. Eng. Chem. Res, 49:
7631–7637.
41. Jochen W, Sylvia G, Michael D, and Julian M. (2009). Nanostructured Encapsulation Systems: Food
Antimicrobials. Global Issues in Food Science and Technology ISBN 9780123741240, 425-479.
42. Lambert R. J. W, Skandamis P. N, Coote P. J, and Nychas G. J. E. (2001). A study of the minimum inhibitory
concentration and mode of action of oregano essential oil, thymol and carvacrol. J. Appl. Micro- biol, 91:
453–462.
43. Leal-Calderon, F, Thivilliers, F, and Schmitt, V. (2007). Structured emulsions. Curr. Opin. Colloid Interface Sci,
12(4–5), 206–212.
44. Lee K. G. and Shibamoto T. (2001). Antioxidant activities of volatile components isolated from Eucalyptus
species. Journal of the Science of Food and Agriculture, 81: 1573–1579.
45. Liu W, Sun D, Li C, Liu Q, and Xu J. (2006). Formation and stability of paraffin oil-in-water nano-emulsions
prepared by the emulsion inversion point method. Journal of Colloid and Interface Science, 303: 557-563.
46. Madhu G. L. and K. A. Prabhu (1985 a). Immobilization of dextranase on Bentonite. Enzyme Microb. Technol,
7: 279–282.
47. March J. (1968). Advanced organic chemistry: Reactions, Mechanisms and Structure Hill, Kogakusha, Tokyo,
Japan, pp. 63-65.
48. McClements D. J. (1999). Emulsion rheology. In: Food emulsions principles, practice and techniques. CRC Press,
Washington, DC, pp 235–266.
49. McClements D. J. (2005). Emulsion stability. In: Food emulsions: principles, practices, and techniques, 2 nd edn.
CRC Press, New York, pp 269–339.
50. McClements D. J. and Li Y. (2010). Structured emulsion-based delivery systems: controlling the digestion and
release of lipophilic food components. Advances in Colloid and Interface Science, 59(2): 213-228.
51. McClements D. J, Decker E. A. and Weiss J. (2007). Emulsion-based delivery systems for lipophilic bioactive
components. Journal of Food Science, 72(8): 109-124.
52. McClements J. D. (1999). Food Emulsions: Principles, Practices and Techniques. CRC Press, Boca Raton,
Florida. Nuchuchua, O, Sakulku, U, Uawongyart, N, Puttipipatkhachorn, S, Soottitantawat.
53. Mitsuda H, Yuasumoto K, and Iwami K. (1996). Antioxidation action of indole compounds during the
autoxidation of linoleic acid. Eiyo to Shokuryo, 19: 210-214.
54. Mourtzinos N, Kalogeropoulos S. E, Papadakis K. K. and Karathanos V. T. (2008). Encapsulation of

Nutraceutical Monoterpenes in B-Cyclodextrin and Modified Starch. Journal of Food Science, 73(1): 89-94.
55. Ochomogo M, and Monsalve-Gonzalez A. (2009). Natural fl avor enhancement compositions for food emulsions.
U.S. Patent 2009/0196972 A1. Clorox Co, Oakland
56. Osawa T, and Namiki M. (1981). A novel type of antioxidant isolated from leaf wax of eucalyptus leaves.
Agricultural and Biological Chemistry, 45: 735–739.
57. Ou B, Huang D, Woodill-Hampsch M, Flanagan J. A, and Deemer E. K. (2002). Analysis of antioxidant activities
of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant
power (FRAP) assays: a comparative study. J. Agric. Food Chem, 50: 3122.
58. Oyaizu M. (1986). Studies on products of browning reaction: antioxidative activity of products of browning
reaction. JPN. J. Nutr, 44: 307-315.
59. Politeo O, Jukic M, and Milos M. (2007). Chemical composition and antioxidant capacity of free volatile
aglycones from basil (Ocimum basilicum L.) compared with its essential oil. Food Chemistry, 101: 379–385.
60. Prieto P, Pineda M, and Aguilar M. (1999). Spectrophotometric quantitation of antioxidant capacity through the
formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal.
Biochem. 269: 337–341.
61. Ravi K. M. N. (2000). Nano and microparticles as controlled drug delivery devices, Journal of Pharmacy and
Pharmaceutical Sciences, 3(2): 234–258.
62. Regiane V. B. F, Soraia V. B, and Diego A. B. (2013). Influence of spray drying operating conditions on
microencapsulated rosemary essential oil properties. Ciênc. Tecnol. Aliment, Campinas, 33(1): 171-178.
63. Rima K, Consolate N, Hao Z, and Wei C. (2013). Evaluation of antimicrobial and antioxidant activities of Syrian
sumac fruit extract. Journal of Natural Products, 6: 96-102.
64. Rima K, Consolate N, Hao Z, and Wei C. (2013). Evaluation of antimicrobial and antioxidant activities of Syrian
sumac fruit extract. Journal of Natural Products, 6: 96-102
65. Robert P, Tamara G, Nalda R, Elena S, Jorge C, and Carmen S. (2010). Encapsulation of polyphenols and
anthocyanins from pomegranate (Punica granatum) by spray drying. International Journal of Food Science and
Technology, 45(7): 1386-1394.
66. Ruch R. J, Cheng S. J, and Klaunig J. E. (1989). Prevention of cytotoxicity and inhibition of intracellular
communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis, 10: 1003-1008.
67. Sahaa M. R, Hasana S. M. R, Aktera R, Hossaina M. M, Alamb M. S, Alam M. A, and Mazumderc M. E. H.

(2008). In vitro free radical scavenging activity of methanol extract of the leaves of Mimusops elengilinn. Bangl.
J. Vet. Med, 6 (2): 197–202.
68. Sikkema J, Debont J. A. M, and Poolman B. (1995). Mechanisms of membrane toxicity of hydrocarbons.
Microbiological Reviews 59: 201–222.
69. Sliwinski E. L, Lavrijsen B. W. M, Vollenbroek J. M, van der Stege H. J, van Boekel M. A. J. S. and Wouters J.
T. M. (2003). Effects of spray drying on physicochemical properties of milk protein-stabilised emulsions.
Colloids and Surfaces B: Biointerfaces, 31(1-4): 219-229.
70. Spicer P. T. (2004). Cubosomes: bicontinuous liquid crystalline nanoparticles. In: Nalwa H (ed) Encyclopedia of
nanoscience and nanotechnology. Marcel Dekker, New York, pp 881–892.
71. Tan C. P. (2005). Effect of polyglycerol esters of fatty acids on physicochemical properties and stability of
-carotene nanodispersions prepared by emulsification/evaporation method. J. Sci. Food Agric, 85: 121–126.
72. Teissedre P. L, and Waterhouse A. L. (2000). Inhibition of oxidation of human low-density lipoproteins by
phenolic substances in different essential oils varieties. Journal of Agricultural and Food Chemistry,
48: 3801–3805.
73. Toure A, Xiaoming Z, Jia C. and Zhijian D. (2007). Microencapsulation and oxidative stability of ginger essential
oil in maltodextrin/whey protein isolate (md/wpi). International Journal of Dairy Science, 2(4): 387-392.
74. Ultee A, Bennik M. H. J, and Moezelaar R. (2002). Thepheno-lic hydroxyl group of carvacrolis essential for
action against the food- borne pathogen Bacilluscereus. Appl. Environ. Microbiol, 68, 1561–1568.
75. Ultee A, Kets E. P. W, Alberda M, Hoekstra F. A, and Smid E. J. (2000). Adaptation of the food-borne pathogen
Bacilluscereus tocarvacrol. Arch. Microbiol, 174: 233–238.
76. Weiss J, Gaysinksy S, Davidson M, Mcclements J. (2009). Nanostructured encapsulation systems: food
antimicrobials. In: Barbosa-C‫ل‬novas, G.V, Mortimer, A, Lineback, D, Spiess, W, Buckle, K. (Eds.), IUFoST
World Congress Book: Global Issues in Food Science and Technology. Elsevier Inc, Amsterdam.
77. Wettasinghe M. and Shahidi F. (1999). Evening primrose meal: a source of natural antioxidants and scavenger of
hydrogen peroxide and oxygen-derived free radicals. J. Agric. Food Chem, 49: 1801–1812.
78. Xu Q. Y, Nakajima M, Binks B. P. (2005). Preparation of particle-stabilized oil-in-water emulsions with the
microchannel emulsifi cation method. Colloids Surf A Physicochem, Eng. Asp, 262: 94–100.
79. Yuan Y, GAO Y, Mao L, and Zhao J. (2008). Optimisation of conditions for the preparation of b-carotene
nanoemulsions using response surface methodology. Food Chemistry, 107: 1300-1306.
80. Zeller B. L, Saleeb F. Z. and Ludescher R. D. (1998). Trends in development of porous carbohydrate food
ingredients for use in flavor encapsulation. Trends in Food Science and Technology, 9(11-12): 389-394.
View publication stats

Nanoemulsion and Nanoencapsulation of Something

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Nanoemulsion and Nanoencapsulation of Something

Uploaded by

Copyright:

Available Formats

See discussions, stats, and author proﬁles for this publication at: https://www.researchgate.

Nanoencapsulation and nanoemulsion of bioactive compounds to enhance

Article · December 2014

Khaled Fahmy Mahmoud Khaled M A Ramadan

SEE PROFILE SEE PROFILE

natural additives View project

Nanotechnology and Biotechnology View project

The user has requested enhancement of the downloaded ﬁle.

NANOENCAPSULATION AND NANOEMULSION OF BIOACTIVE COMPOUNDS TO

KHALED F. M1, KHALED M. A. RAMADAN2 & I. S. ASHOUSH3

KEYWORDS: Nano-Emulsion, Nano-Encapsulation, Bioactive Compounds, Thymol, Carvacrol, Eugenol, Linalool

MATERIALS AND METHODS

Total Antioxidant Capacity as Equivalents of α-Tocopheroel

% DPPH radical scavenging activity = ((Ac-As)/Ac) ×100

Scavenging of Hydrogen Peroxide

% scavenging of hydrogen peroxide effect = (As/Ac -1) ×100

Antioxidant Activity in a Linoleic Acid System

Antioxidant activity %= 100 - [(absorbance of sample / absorbance of control) ×100]

All tests and analyses were run in triplicate and averaged

Preparation of Bioactive Nanoemulsions

Determination of Nano-Encapsulation Efficiency (NE) of the Clove Powder

Oxidation Study of Bioactive Compounds Encapsulated forms by DSC

Application and Preparation of Jelly

Phase Solubility Studies

RESULTS AND DISCUSSIONS

Table 2: The Antioxidant Capacity of Thymol, Carvacrol, Linalool and Eugenol

Table 4: The Antioxidant Activity of Thymol, Carvacrol, Linalool and

Characterization of Nanoemulsion and Nanoencapsulation of Bioactive Compounds

Nano-Encapsulation Yield (EY)

Figure 1: Nano-Encapsulation Yield of of Thymol, Carvacrol, Linalool and Eugenol in Dextran

Encapsulation Efficiency (%EE)

Figure 2: Nano-Encapsulation Efficiency of Thymol, Carvacrol, Linolool and Eugenol in Dextran

Solubility of the Eugenol Nano-Encapsulated

Figure 4: Time Related Release of Dextran Nano-Encapsulated Eugenol in Water

Thermal Stability of Nano Eugenol Capsules DSC (Differential Scanning Coliremetry)

Figure 5: DSC Thermogram of Eugenol before (a) and After Nanoencapsulated

Figure 6: Morphology of Eugenol after Nanoemulsion (a) and Nanoencapsulation

Storage Stability of Nano-Encapsulated Eugenol

Table 5: Antioxidant Activity of Nano-Encapsulated Eugenol (Dex/T20) in Linolic

Sensory Evaluation of Jelly

Table 6: Sensory Evaluation of Jelly Prepared with Different Levels of

Table 7: The Microbiological Characteristics of the Jelly with or

Control: is jelly without antioxidant; cfu g-1: colony forming units/g

2. Ahmed M. G, Satish K. B. P, Kiran K. G. B. (2010). Formulation and evaluation of gastric-mucoadhesive drug

9. Baranauskiene R, Venskutonis P. R, Dewettinck K. and Verhé R. (2006). Properties of oregano

Conference Centre (BCFCC), Sher-e-Bangla Nagar, Dhaka, Bangladesh. 9-15.

54. Mourtzinos N, Kalogeropoulos S. E, Papadakis K. K. and Karathanos V. T. (2008). Encapsulation of

U.S. Patent 2009/0196972 A1. Clorox Co, Oakland

67. Sahaa M. R, Hasana S. M. R, Aktera R, Hossaina M. M, Alamb M. S, Alam M. A, and Mazumderc M. E. H.

You might also like